FXII and polyP bind to the surface of stimulated platelets. (A-B) Washed human platelets were incubated with DL488-labeled FXII and DAPI to detect platelet-derived polyP and were left unstimulated (unstim) or stimulated with 100 ng/mL of CVX and 100 nM thrombin (cvx/th) or with 100 ng/mL of CVX and 20 μM TRAP-6 (cvx/T6), with or without 5 mM GPRP, for 45 minutes at ambient temperature before analyzing DL488-FXII-positive cells (A) and DAPI-positive cells (B) by flow cytometry. **P < .01, ****P < .0001 vs unstim platelets; †P < .05, ††P < .01, ††††P < .0001 cvx/th-stimulated platelets vs cvx/th + GPRP. (C-E) Washed platelets (5 × 107/mL) were activated with 20 μg/mL of collagen and 100 nM thrombin in the presence of DL488-FXII (green) and DAPI (blue; seen in panel E) and stained using AF647-annexin V to detect PS (red). Images represent 3-dimensional render of z-stacks. Scale bars represent 5 μm. Representative images of 3 separate experiments. (F) Platelets (1.5 × 108/mL final concentration) were activated with 100 μg/mL of collagen and 100 μM TRAP-6 in the presence of CaCl2 before adding to plasma clots (50%) in the presence of DyLight 550 (DL550)-labeled fibrinogen (120 nM; orange), DL488-FXII (365 nM; green), DAPI (20 μg/mL; light gray), and AF647-annexin V (1/20; red). Thrombin (24 nM) and CaCl2 (10 mM) were added, and clots were allowed to form for 2 hours. The image is representative of 3 separate experiments and displays a 3-dimensional render of a z-stack. Scale bar represents 10 μm. Images in C-F were obtained using a Zeiss LSM710 confocal microscope with a ×63/1.40 oil immersion objective and were analyzed using Zen 2012 software. DIC, differential interference contrast; MFI, median fluorescence intensity.
